Crystalline silica-induced pulmonary inflammation and autoimmunity in mature adult NZBW/f1 mice: age-related sensitivity and impact of omega-3 fatty acid intervention.

OBJECTIVE: Occupational exposure to respirable crystalline silica (cSiO2) has been linked to lupus development. Previous studies in young lupus-prone mice revealed that intranasal cSiO2 exposure triggered autoimmunity, preventable with docosahexaenoic acid (DHA). This study explores cSiO2 and DHA effects in mature lupus-prone adult mice, more representative of cSiO2-exposed worker age. METHODS: Female NZBWF1 mice (14-week old) were fed control (CON) or DHA-supplemented diets. After two weeks, mice were intranasally instilled saline (VEH) or 1 mg cSiO2 weekly for four weeks. Cohorts were then analyzed 1- and 5-weeks postinstillation for lung inflammation, cell counts, chemokines, histopathology, B- and T-cell infiltration, autoantibodies, and gene signatures, with results correlated to autoimmune glomerulonephritis onset. RESULTS: VEH/CON mice showed no pathology. cSiO2/CON mice displayed significant ectopic lymphoid tissue formation in lungs at 1 week, increasing by 5 weeks. cSiO2/CON lungs exhibited elevated cellularity, chemokines, CD3+ T-cells, CD45R + B-cells, IgG + plasma cells, gene expression, IgG autoantibodies, and glomerular hypertrophy. DHA supplementation mitigated all these effects. DISCUSSION: The mature adult NZBWF1 mouse used here represents a life-stage coincident with immunological tolerance breach and one that more appropriately represents the age (20-30 yr) of cSiO2-exposed workers. cSiO2-induced robust pulmonary inflammation, autoantibody responses, and glomerulonephritis in mature adult mice, surpassing effects observed previously in young adults. DHA at a human-equivalent dosage effectively countered cSiO2-induced inflammation/autoimmunity in mature mice, mirroring protective effects in young mice. CONCLUSION: These results highlight life-stage significance in this preclinical lupus model and underscore omega-3 fatty acids' therapeutic potential against toxicant-triggered autoimmune responses.

Dietary docosahexaenoic acid supplementation inhibits acute pulmonary transcriptional and autoantibody responses to a single crystalline silica exposure in lupus-prone mice.

Introduction: Workplace exposure to respirable crystalline silica (cSiO2) has been epidemiologically linked to lupus. Consistent with this, repeated subchronic intranasal cSiO2 instillation in lupus-prone NZBWF1 mice induces inflammation-/autoimmune-related gene expression, ectopic lymphoid tissue (ELT), autoantibody (AAb) production in the lung within 5 to 13 wk followed systemic AAb increases and accelerated onset and progression of glomerulonephritis within 13 to 17 wk. Interestingly, dietary docosahexaenoic acid (DHA) supplementation suppresses these pathologic effects, but the underlying molecular mechanisms remain unclear. Methods: This study aimed to test the hypothesis that dietary DHA supplementation impacts acute transcriptional and autoantibody responses in the lungs of female NZBWF1 mice 1 and 4 wk after a single high-dose cSiO2 challenge. Groups of mice were initially fed a control (Con) diet or a DHA-containing diet (10 g/kg). Cohorts of Con- and DHA-fed were subjected to a single intranasal instillation of 2.5 mg cSiO2 in a saline vehicle (Veh), while a Con-fed cohort was instilled with Veh only. At 1 and 4 wk post-instillation (PI), we compared cSiO2's effects on innate-/autoimmune-related gene expression and autoantibody (AAb) in lavage fluid/lungs of Con- and DHA-fed mice and related these findings to inflammatory cell profiles, histopathology, cell death, and cytokine/chemokine production. Results: DHA partially alleviated cSiO2-induced alterations in total immune cell and lymphocyte counts in lung lavage fluid. cSiO2-triggered dead cell accumulation and levels of inflammation-associated cytokines and IFN-stimulated chemokines were more pronounced in Con-fed mice than DHA-fed mice. Targeted multiplex transcriptome analysis revealed substantial upregulation of genes associated with autoimmune pathways in Con-fed mice in response to cSiO2 that were suppressed in DHA-fed mice. Pathway analysis indicated that DHA inhibited cSiO2 induction of proinflammatory and IFN-regulated gene networks, affecting key upstream regulators (e.g., TNFα, IL-1ß, IFNAR, and IFNγ). Finally, cSiO2-triggered AAb responses were suppressed in DHA-fed mice. Discussion: Taken together, DHA mitigated cSiO2-induced upregulation of pathways associated with proinflammatory and IFN-regulated gene responses within 1 wk and reduced AAb responses by 4 wk. These findings suggest that the acute short-term model employed here holds substantial promise for efficient elucidation of the molecular mechanisms through which omega-3 PUFAs exert protective effects against cSiO2-induced autoimmunity.

Investigating the Combined Toxicity of Cu(II) and Carbon Monoxide (CO); Cellular CO Delivery Using a Cu(II) Flavonolato Complex.

Carbon monoxide (CO) delivery molecules are of significant current interest as potential therapeutics, including for anticancer applications. A recent approach toward generating new types of materials-based anticancer agents involves combining the Fenton reactivity of a redox active metal ion with CO delivery. However, small molecule examples of these types of entities have not been systematically studied to evaluate the combined effect on cellular toxicity. Herein we describe a Cu(II) flavonolato complex which produces anticancer effects through a combination of copper-mediated reactive oxygen species (ROS) generation and light-induced flavonol CO release. Confocal microscopy studies provide evidence of enhanced flavonol uptake in the copper flavonolato system relative to the free flavonol, which leads to an increased amount of CO delivery within cells. Importantly, this work demonstrates that a metal flavonolato species can be used to produce enhanced toxicity effects resulting from both metal ion-induced Fenton reactivity and increased cellular uptake of a flavonol CO donor.

Basal Diet Fed to Recipient Mice Was the Driving Factor for Colitis and Colon Tumorigenesis, despite Fecal Microbiota Transfer from Mice with Severe or Mild Disease.

Consumption of the total Western diet (TWD) in mice has been shown to increase gut inflammation, promote colon tumorigenesis, and alter fecal microbiome composition when compared to mice fed a healthy diet, i.e., AIN93G (AIN). However, it is unclear whether the gut microbiome contributes directly to colitis-associated CRC in this model. The objective of this study was to determine whether dynamic fecal microbiota transfer (FMT) from donor mice fed either the AIN basal diet or the TWD would alter colitis symptoms or colitis-associated CRC in recipient mice, which were fed either the AIN diet or the TWD, using a 2 × 2 factorial experiment design. Time-matched FMT from the donor mice fed the TWD did not significantly enhance symptoms of colitis, colon epithelial inflammation, mucosal injury, or colon tumor burden in the recipient mice fed the AIN diet. Conversely, FMT from the AIN-fed donors did not impart a protective effect on the recipient mice fed the TWD. Likewise, the composition of fecal microbiomes of the recipient mice was also affected to a much greater extent by the diet they consumed than by the source of FMT. In summary, FMT from the donor mice fed either basal diet with differing colitis or tumor outcomes did not shift colitis symptoms or colon tumorigenesis in the recipient mice, regardless of the basal diet they consumed. These observations suggest that the gut microbiome may not contribute directly to the development of disease in this animal model.

Dietary Supplementation with Black Raspberries Altered the Gut Microbiome Composition in a Mouse Model of Colitis-Associated Colorectal Cancer, although with Differing Effects for a Healthy versus a Western Basal Diet.

Black raspberries (BRB) are rich in anthocyanins with purported anti-inflammatory properties. However, it is not known whether dietary supplementation would ameliorate Western-diet enhanced gut inflammation and colon tumorigenesis. We employed a mouse model of colitis-associated colorectal cancer (CAC) to determine the effects of dietary supplementation with 5 to 10% (w/w) whole, freeze-dried BRB in male C57BL/6J mice fed either a standard healthy diet (AIN93G) or the total Western diet (TWD). In a pilot study, BRB suppressed colitis and colon tumorigenesis while also shifting the composition of the fecal microbiome in favor of taxa with purported health benefits, including Bifidobacterium pseudolongum. In a follow-up experiment using a 2 × 2 factorial design with AIN and TWD basal diets with and without 10% (w/w) BRB, supplementation with BRB reduced tumor multiplicity and increased colon length, irrespective of the basal diet, but it did not apparently affect colitis symptoms, colon inflammation or mucosal injury based on histopathological findings. However, BRB intake increased alpha diversity, altered beta diversity and changed the relative abundance of Erysipelotrichaceae, Bifidobacteriaceae, Streptococcaceae, Rikenellaceae, Ruminococcaceae and Akkermansiaceae, among others, of the fecal microbiome. Notably, changes in microbiome profiles were inconsistent with respect to the basal diet consumed. Overall, these studies provide equivocal evidence for in vivo anti-inflammatory effects of BRB on colitis and colon tumorigenesis; yet, BRB supplementation led to dynamic changes in the fecal microbiome composition over the course of disease development.

Comparative effects of human-equivalent low, moderate, and high dose oral prednisone intake on autoimmunity and glucocorticoid-related toxicity in a murine model of environmental-triggered lupus.

Autoimmune diseases can be triggered by environmental toxicants such as crystalline silica dust (cSiO2). Here, we characterized the dose-dependent immunomodulation and toxicity of the glucocorticoid (GC) prednisone in a preclinical model that emulates onset and progression of cSiO2-triggered lupus. Two cohorts of 6-wk-old female NZBWF1 mice were fed either control AIN-93G diet or one of three AIN-93G diets containing prednisone at 5, 15, or 50 mg/kg diet which span human equivalent oral doses (HED) currently considered to be low (PL; 5 mg/d HED), moderate (PM; 14 mg/d HED), or high (PH; 46 mg/d HED), respectively. At 8 wk of age, mice were intranasally instilled with either saline vehicle or 1 mg cSiO2 once weekly for 4 wk. The experimental plan was to 1) terminate one cohort of mice (n=8/group) 14 wk after the last cSiO2 instillation for pathology and autoimmunity assessment and 2) to maintain a second cohort (n=9/group) to monitor glomerulonephritis development and survival. Mean blood concentrations of prednisone's principal active metabolite, prednisolone, in mice fed PL, PM, and PH diets were 27, 105, 151 ng/ml, respectively, which are consistent with levels observed in human blood ≤ 12 h after single bolus treatments with equivalent prednisone doses. Results from the first cohort revealed that consumption of PM, but not PL diet, significantly reduced cSiO2-induced pulmonary ectopic lymphoid structure formation, nuclear-specific AAb production, inflammation/autoimmune gene expression in the lung and kidney, splenomegaly, and glomerulonephritis in the kidney. Relative to GC-associated toxicity, PM diet, but not PL diet, elicited muscle wasting, but these diets did not affect bone density or cause glucosuria. Importantly, neither PM nor PL diet improved latency of cSiO2-accelerated death. PH-fed mice in both cohorts displayed robust GC-associated toxicity including body weight loss, reduced muscle mass, and extensive glucosuria 7 wk after the final cSiO2 instillation requiring their early removal from the study. Taken together, our results demonstrate that while moderate doses of prednisone can reduce important pathological endpoints of cSiO2-induced autoimmunity in lupus-prone mice, such as upstream ectopic lymphoid structure formation, these ameliorative effects come with unwanted GC toxicity, and, crucially, none of these three doses extended survival time.

Flavonol-Based Carbon Monoxide Delivery Molecule with Endoplasmic Reticulum, Mitochondria, And Lysosome Localization.

Light-triggered carbon monoxide (CO) delivery molecules are of significant current interest for evaluating the role of CO in biology and as potential therapeutics. Herein we report the first example of a metal free CO delivery molecule that can be tracked via confocal microscopy at low micromolar concentrations in cells prior to CO release. The NEt2-appended extended flavonol (4) localizes to the endoplasmic reticulum, mitochondria, and lysosomes. Subcellular localization of 4 results in CO-induced toxicity effects that are distinct as compared to a nonlocalized analog. Anti-inflammatory effects of 4, as measured by TNF-α suppression, occur at the nanomolar level in the absence of CO release, and are enhanced with visible-light-induced CO release. Overall, the highly trackable nature of 4 enables studies of the biological effects of both a localized flavonol and CO release at low micromolar to nanomolar concentrations.

Comparing mRNA and sncRNA profiles during the maternal-to-embryonic transition in bovine IVF and scNT embryos†.

Production of embryos with high developmental competence by somatic cell nuclear transfer (scNT) is far less efficient than for in vitro fertilized (IVF) embryos, likely due to an accumulation of errors in genome reprogramming that results in aberrant expression of RNA transcripts, including messenger RNAs (mRNA) and, possibly, microRNAs (miRNA). Thus, our objectives were to use RNAseq to determine the dynamics of mRNA expression in early developing scNT and IVF embryos in the context of the maternal-to-embryonic transition (MET) and to correlate apparent transcriptional dysregulation in cloned embryos with miRNA expression profiles. Comparisons between scNT and IVF embryos indicated large scale transcriptome differences, which were most evident at the 8-cell and morula stages for genes associated with biological functions critical for the MET. For two miRNAs previously identified as differentially expressed in scNT morulae, miR-34a and miR-345, negative correlations with some predicted mRNA targets were apparent, though not widespread among the majority of predicted targets. Moreover, although large-scale aberrations in expression of mRNAs were evident during the MET in cattle scNT embryos, these changes were not consistently correlated with aberrations in miRNA expression at the same developmental stage, suggesting that other mechanisms controlling gene expression may be involved.

Dynamics of small non-coding RNAs in bovine scNT embryos through the maternal-to-embryonic transition†.

The efficiency of somatic cell nuclear transfer (scNT) for production of viable offspring is relatively low as compared to in vitro fertilization (IVF), presumably due to deficiencies in epigenetic reprogramming of the donor cell genome. Such defects may also involve the population of small non-coding RNAs (sncRNAs), which are important during early embryonic development. The objective of this study was to examine dynamic changes in relative abundance of sncRNAs during the maternal-to-embryonic transition (MET) in bovine embryos produced by scNT as compared to IVF by using RNA sequencing. When comparing populations of miRNA in scNT versus IVF embryos, only miR-2340, miR-345, and miR34a were differentially expressed in morulae, though many more miRNAs were differentially expressed when comparing across developmental stages. Also of interest, distinct populations of piwi-interacting like RNAs (pilRNAs) were identified in bovine embryos prior to and during embryonic genome activation (EGA) as compared bovine embryos post-EGA and differentiated cells. Overall, sncRNA sequencing analysis of preimplantation embryos revealed largely similar profiles of sncRNAs for IVF and scNT embryos at the 2-cell, 8-cell, morula, and blastocyst stages of development. However, these sncRNA profiles, including miRNA, piRNA, and tRNA fragments, were notably distinct prior to and after completion of the MET.

The Western Dietary Pattern Combined with Vancomycin-Mediated Changes to the Gut Microbiome Exacerbates Colitis Severity and Colon Tumorigenesis.

Previous work by our group using a mouse model of inflammation-associated colorectal cancer (CAC) showed that the total Western diet (TWD) promoted colon tumor development. Others have also shown that vancomycin-mediated changes to the gut microbiome increased colorectal cancer (CRC). Therefore, the objective of this study was to determine the impact of vancomycin on colon tumorigenesis in the context of a standard mouse diet or the TWD. A 2 × 2 factorial design was used, in which C57Bl/6J mice were fed either the standard AIN93G diet or TWD and with vancomycin in the drinking water or not. While both the TWD and vancomycin treatments independently increased parameters associated with gut inflammation and tumorigenesis compared to AIN93G and plain water controls, mice fed the TWD and treated with vancomycin had significantly increased tumor multiplicity and burden relative to all other treatments. Vancomycin treatment significantly decreased alpha diversity and changed the abundance of several taxa at the phylum, family, and genus levels. Conversely, basal diet had relatively minor effects on the gut microbiome composition. These results support our previous research that the TWD promotes colon tumorigenesis and suggest that vancomycin-induced changes to the gut microbiome are associated with higher tumor rates.

Rapid Induction of Pulmonary Inflammation, Autoimmune Gene Expression, and Ectopic Lymphoid Neogenesis Following Acute Silica Exposure in Lupus-Prone Mice.

Occupational exposure to crystalline silica (cSiO2) is etiologically associated with systemic lupus erythematosus (lupus) and other autoimmune diseases. cSiO2's autoimmune effects in humans can be mimicked chronically in female lupus-prone NZBWF1 mice following repeated exposure to the particle. However, the immediate and short-term effects of cSiO2 in this widely used model of autoimmune disease are not well-understood. In the present study, we tested the hypothesis that a single acute cSiO2 dose triggers early presentation of cellular, histopathological, transcriptomic, and protein biomarkers of inflammation and autoimmunity in lupus-prone mice. Eight-week old female NZBWF1 mice were intranasally instilled once with 2.5 mg cSiO2 or saline vehicle and necropsied at 1, 7, 14, 21, and 28 d post-instillation (PI). Analyses of bronchoalveolar lavage fluid (BALF) and lung tissue revealed that by 7 d PI, acute cSiO2 exposure persistently provoked: (i) robust recruitment of macrophages, neutrophils, and lymphocytes into the alveoli, (ii) cell death as reflected by increased protein, double-stranded DNA, and lactate dehydrogenase activity, (iii) elevated secretion of the cytokines IL-1α, IL-1ß, IL-18, TNF-α, IL-6, MCP-1, and B cell activation factor (BAFF), and (iv) upregulation of genes associated with chemokines, proinflammatory cytokines, lymphocyte activation, and type I interferon signaling. The appearance of these endpoints was subsequently followed by the emergence in the lung of organized CD3+ T cells (14 d PI) and CD45R+ B cells (21 d PI) that were indicative of ectopic lymphoid structure (ELS) development. Taken together, acute cSiO2 exposure triggered a rapid onset of autoimmune disease pathogenesis that was heralded in the lung by unresolved inflammation and cell death, proinflammatory cytokine production, chemokine-driven recruitment of leukocytes, an interferon response signature, B and T cell activation, and ELS neogenesis. This short-term murine model provides valuable new insight into potential early mechanisms of cSiO2-induced lupus flaring and, furthermore, offers a rapid venue for evaluating interventions against respirable particle-triggered inflammation and autoimmunity.

Silica Induction of Diverse Inflammatory Proteome in Lungs of Lupus-Prone Mice Quelled by Dietary Docosahexaenoic Acid Supplementation.

Repeated short-term intranasal instillation of lupus-prone mice with crystalline silica (cSiO2) induces inflammatory gene expression and ectopic lymphoid neogenesis in the lung, leading to early onset of systemic autoimmunity and rapid progression to glomerulonephritis. These responses are suppressed by dietary supplementation with the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA). Here, we tested the hypothesis that dietary DHA supplementation suppresses cSiO2-induced inflammatory proteins in bronchoalveolar alveolar lavage fluid (BALF) and plasma of lupus-prone mice. Archived tissue fluid samples were used from a prior investigation in which 6 wk-old lupus-prone female NZBWF1 mice were fed isocaloric diets containing 0 or 10 g/kg DHA for 2 wks and then intranasally instilled with 1 mg cSiO2 or vehicle once weekly for 4 wks. Cohorts were terminated at 1, 5, 9 or 13 wk post-instillation (PI). BALF and plasma from each cohort were analyzed by high density multiplex array profiling of 200 inflammatory proteins. cSiO2 time-dependently induced increases in the BALF protein signatures that were highly reflective of unresolved lung inflammation, although responses in the plasma were much less robust. Induced proteins in BALF included chemokines (e.g., MIP-2, MCP-5), enzymes (e.g., MMP-10, granzyme B), adhesion molecules (e.g., sE-selectin, sVCAM-1), co-stimulatory molecules (e.g., sCD40L, sCD48), TNF superfamily proteins (e.g., sTNFRI, sBAFF-R), growth factors (e.g., IGF-1, IGFBP-3), and signal transduction proteins (e.g., MFG-E8, FcgRIIB), many of which were blocked or delayed by DHA supplementation. The BALF inflammatory proteome correlated positively with prior measurements of gene expression, pulmonary ectopic lymphoid tissue neogenesis, and induction of autoantibodies in the lungs of the control and treatment groups. Ingenuity Pathway Analysis (IPA) revealed that IL-1ß, TNF-α, and IL-6 were among the top upstream regulators of the cSiO2-induced protein response. Furthermore, DHA's effects were associated with downregulation of cSiO2-induced pathways involving i) inhibition of ARE-mediated mRNA decay, ii) bacterial and viral pattern recognition receptor activation, or iii) TREM1, STAT3, NF-κB, and VEGF signaling and with upregulation of PPAR, LXR/RXR and PPARα/RXRα signaling. Altogether, these preclinical findings further support the contention that dietary DHA supplementation could be applicable as an intervention against inflammation-driven autoimmune triggering by cSiO2 or potentially other environmental agents.

Requisite Omega-3 HUFA Biomarker Thresholds for Preventing Murine Lupus Flaring.

Lupus is a systemic autoimmune disease typified by uncontrolled inflammation, disruption of immune tolerance, and intermittent flaring - events triggerable by environmental factors. Preclinical and clinical studies reveal that consumption of the marine ω-3 highly unsaturated fatty acids (HUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) might be used as a precision nutrition intervention to lessen lupus symptoms. The anti-inflammatory and pro-resolving effects of ω-3 HUFAs are inextricably linked to their presence in membrane phospholipids. The ω-3 HUFA score, calculated as [100 × (ω-3 HUFAs/(ω-3 HUFAs + ω-6 HUFAs))] in red blood cells (RBCs), and the Omega-3 Index (O3I), calculated as [100 × ((DHA+EPA)/total fatty acids)] in RBCs, are two biomarkers potentially amenable to relating tissue HUFA balance to clinical outcomes in individuals with lupus. Using data from three prior preclinical DHA supplementation studies, we tested the hypothesis that the ω-3 HUFA score and the O3I inversely correlate with indicators of autoimmune pathogenesis in the cSiO2-triggered lupus flaring model. The three studies employed both low and high fat rodent diets, as well as more complex diets emulating the U.S. dietary pattern. The ω-3 HUFA scores in RBCs were comparatively more robust than the O3I at predicting HUFA balances in the kidney, liver, spleen, and lung. Importantly, increases in both the ω-3 HUFA score (>40%) and the O3I (>10%) were strongly associated with suppression of cSiO2-triggered (1) expression of interferon-regulated genes, proinflammatory cytokine production, leukocyte infiltration, and ectopic lymphoid structure development in the lung, (2) pulmonary and systemic autoantibody production, and (3) glomerulonephritis. Collectively, these findings identify achievable ω-3 HUFA scores and O3I thresholds that could be targeted in future human intervention studies querying how ω-3 HUFA consumption influences lupus and other autoimmune diseases.

Omega-3 fatty acid intake suppresses induction of diverse autoantibody repertoire by crystalline silica in lupus-prone mice.

Inhalation of crystalline silica (cSiO2) in the workplace is etiologically linked to lupus and other autoimmune diseases. Exposing lupus-prone NZBWF1 mice to respirable cSiO2 unleashes a vicious cycle of inflammation and cell death in the lung that triggers interferon-regulated gene expression, ectopic lymphoid structure (ELS) development, elevation of local and systemic autoantibodies (AAbs), and glomerulonephritis. However, cSiO2-induced inflammation and onset of autoimmunity can be prevented by inclusion of the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) into the diet of these mice. Since cSiO2 both causes cell death and interferes with efferocytosis, secondary necrosis of residual cell corpses might provide a rich and varied autoantigen (AAg) source in the lung. While it is known that the particle induces anti-nuclear and anti-dsDNA AAbs in NZBWF1 mice, the full extent of the cSiO2-induced AAb response relative to specificity and isotype is not yet understood. The purpose of this study was to test the hypotheses that cSiO2 exposure induces a wide spectrum of AAbs in the pulmonary and systemic compartments, and that dietary DHA intervention prevents these changes. Archived tissue fluid samples were obtained from a prior study in which NZBWF1 mice were fed purified isocaloric diets containing no DHA (control) or DHA corresponding calorically to human doses of 2 and 5 g/day. Mice were intranasally instilled with 1 mg cSiO2 or saline vehicle weekly for 4 weeks, then groups euthanized 1, 5, 9, or 13 weeks post-instillation (PI) of the last cSiO2 dose. Bronchoalveolar lavage fluid (BALF) and plasma from each time point were subjected to AAb profiling using a microarray containing 122 AAgs. cSiO2 triggered robust IgG and IgM AAb responses against lupus-associated AAgs, including DNA, histones, ribonucleoprotein, Smith antigen, Ro/SSA, La/SSB, and complement as early as 1 week PI in BALF and 5 weeks PI in plasma, peaking at 9 and 13 weeks PI, respectively. Importantly, cSiO2 also induced AAbs to AAgs associated with rheumatoid arthritis (collagen II, fibrinogen IV, fibrinogen S, fibronectin, and vimentin), Sjögren's syndrome (α-fodrin), systemic sclerosis (topoisomerase I), vasculitis (MPO and PR3), myositis (Mi-2, TIF1-γ, MDA5), autoimmune hepatitis (LC-1), and celiac disease (TTG). cSiO2 elicited comparable but more modest IgA AAb responses in BALF and plasma. cSiO2-induced AAb production was strongly associated with time dependent inflammatory/autoimmune gene expression, ELS development, and glomerulonephritis. AAb responses were dose-dependently suppressed by DHA supplementation and negatively correlated with the ω-3 index, an erythrocyte biomarker of ω-3 content in tissue phospholipids. Taken together, these findings suggest that cSiO2 exposure elicits a diverse multi-isotype repertoire of AAbs, many of which have been reported in individuals with lupus and other autoimmune diseases. Furthermore, induction of this broad AAb spectrum could be impeded by increasing ω-3 tissue content via dietary DHA supplementation.

Development of Triggerable, Trackable, and Targetable Carbon Monoxide Releasing Molecules.

Carbon monoxide (CO) is a gaseous signaling molecule produced in humans via the breakdown of heme in an O2-dependent reaction catalyzed by heme oxygenase enzymes. A long-lived species relative to other signaling molecules (e.g., NO, H2S), CO exerts its physiological effects via binding to low-valent transition metal centers in proteins and enzymes. Studies involving the administration of low doses of CO have shown its potential as a therapeutic agent to produce vasodilation, anti-inflammatory, antiapoptotic, and anticancer effects. In pursuit of developing tools to define better the role and therapeutic potential of CO, carbon monoxide releasing molecules (CORMs) were developed. To date, the vast majority of reported CORMs have been metal carbonyl complexes, with the most well-known being Ru2Cl4(CO)6 (CORM-2), Ru(CO)3Cl(glycinate) (CORM-3), and Mn(CO)4(S2CNMe(CH2CO2H)) (CORM-401). These complexes have been used to probe the effects of CO in hundreds of cell- and animal-based experiments. However, through recent investigations, it has become evident that these reagents exhibit complicated reactivity in biological environments. The interpretation of the effects produced by some of these complexes is obscured by protein binding, such that their formulation is not clear, and by CO leakage and potential redox activity. An additional weakness with regard to CORM-2 and CORM-3 is that these compounds cannot be tracked via fluorescence. Therefore, it is unclear where or when CO release occurs, which confounds the interpretation of experiments using these molecules. To address these weaknesses, our research team has pioneered the development of metal-free CORMs based on structurally tunable extended flavonol or quinolone scaffolds. In addition to being highly controlled, with CO release only occurring upon triggering with visible light (photoCORMs), these CO donors are trackable via fluorescence prior to CO release in cellular environments and can be targeted to specific cellular locations.In the Account, we highlight the development and application of a series of structurally related flavonol photoCORMs that (1) sense characteristics of cellular environments prior to CO release; (2) enable evaluation of the influence of cytosolic versus mitochondrial-localized CO release on cellular bioenergetics; (3) probe the cytotoxicity and anti-inflammatory effects of intracellular versus extracellular CO delivery; and (4) demonstrate that albumin delivery of a photoCORM enables potent anticancer and anti-inflammatory effects. A key advantage of using triggered CO release compounds in these investigations is the ability to examine the effects of the molecular delivery vehicle in the absence and presence of localized CO release, thus providing insight into the independent contributions of CO. Overall, flavonol-based CO delivery molecules offer opportunities for triggerable, trackable, and targetable CO delivery that are unprecedented in terms of previously reported CORMs and, thus, offer significant potential for applications in biological systems.

CO Sense and Release Flavonols: Progress toward the Development of an Analyte Replacement PhotoCORM for Use in Living Cells.

Carbon monoxide (CO) is a signaling molecule in humans. Prior research suggests that therapeutic levels of CO can have beneficial effects in treating a variety of physiological and pathological conditions. To facilitate understanding of the role of CO in biology, molecules that enable fluorescence detection of CO in living systems have emerged as an important class of chemical tools. A key unmet challenge in this field is the development of fluorescent analyte replacement probes that replenish the CO that is consumed during detection. Herein, we report the first examples of CO sense and release molecules that involve combining a common CO-sensing motif with a light-triggered CO-releasing flavonol scaffold. A notable advantage of the flavonol-based CO sense and release motif is that it is trackable via fluorescence in both its pre- and postsensing (pre-CO release) forms. In vitro studies revealed that the PdCl2 and Ru(II)-containing CORM-2 used in the CO sensing step can result in metal coordination to the flavonol, which minimizes the subsequent CO release reactivity. However, CO detection followed by CO release is demonstrated in living cells, indicating that a cellular environment mitigates the flavonol/metal interactions.

Influence of total western diet on docosahexaenoic acid suppression of silica-triggered lupus flaring in NZBWF1 mice.

Lupus is a debilitating multi-organ autoimmune disease clinically typified by periods of flare and remission. Exposing lupus-prone female NZBWF1 mice to crystalline silica (cSiO2), a known human autoimmune trigger, mimics flaring by inducing interferon-related gene (IRG) expression, inflammation, ectopic lymphoid structure (ELS) development, and autoantibody production in the lung that collectively accelerate glomerulonephritis. cSiO2-triggered flaring in this model can be prevented by supplementing mouse diet with the ω-3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA). A limitation of previous studies was the use of purified diet that, although optimized for rodent health, does not reflect the high American intake of saturated fatty acid (SFA), ω-6 PUFAs, and total fat. To address this, we employed here a modified Total Western Diet (mTWD) emulating the 50th percentile U.S. macronutrient distribution to discern how DHA supplementation and/or SFA and ω-6 reduction influences cSiO2-triggered lupus flaring in female NZBWF1 mice. Six-week-old mice were fed isocaloric experimental diets for 2 wks, intranasally instilled with cSiO2 or saline vehicle weekly for 4 wks, and tissues assessed for lupus endpoints 11 wks following cSiO2 instillation. In mice fed basal mTWD, cSiO2 induced robust IRG expression, proinflammatory cytokine and chemokine elevation, leukocyte infiltration, ELS neogenesis, and autoantibody production in the lung, as well as early kidney nephritis onset compared to vehicle-treated mice fed mTWD. Consumption of mTWD containing DHA at the caloric equivalent to a human dose of 5 g/day dramatically suppressed induction of all lupus-associated endpoints. While decreasing SFA and ω-6 in mTWD modestly inhibited some disease markers, DHA addition to this diet was required for maximal protection against lupus development. Taken together, DHA supplementation at a translationally relevant dose was highly effective in preventing cSiO2-triggered lupus flaring in NZBWF1 mice, even against the background of a typical Western diet.

Food matrix and the microbiome: considerations for preclinical chronic disease studies.

Animal models of chronic disease are continuously being refined and have evolved with the goal of increasing the translation of results to human populations. Examples of this progress include transgenic models and germ-free animals conventionalized with human microbiota. The gut microbiome is involved in the etiology of several chronic diseases. Therefore, consideration of the experimental conditions that may affect the gut microbiome in preclinical disease is very important. Of note, diet plays a large role in shaping the gut microbiome and can be a source of variation between animal models and human populations. Traditionally, nutrition researchers have focused on manipulating the macronutrient profile of experimental diets to model diseases such as metabolic syndrome. However, other dietary components found in human foods, but not in animal diets, can have sizable effects on the composition and metabolic capacity of the gut microbiome and, as a consequence, manifestation of the chronic disease being modeled. The purpose of this review is to describe how food matrix food components, including diverse fiber sources, oxidation products from cooking, and dietary fat emulsifiers, shape the composition of the gut microbiome and influence gut health.

Consumption of the Total Western Diet Promotes Colitis and Inflammation-Associated Colorectal Cancer in Mice.

Consumption of a Western type diet is a known risk factor for colorectal cancer. Our group previously developed the total Western diet (TWD) for rodents with energy and nutrient profiles that emulate a typical Western diet. In this study, we tested the hypothesis that consumption of the TWD would enhance colitis, delay recovery from gut injury and promote colon tumorigenesis. In multiple experiments using the azoxymethane + dextran sodium sulfate or ApcMin/+ mouse models of colitis-associated colorectal carcinogenesis (CAC), we determined that mice fed TWD experienced more severe and more prolonged colitis compared to their counterparts fed the standard AIN93G diet, ultimately leading to markedly enhanced colon tumorigenesis. Additionally, this increased tumor response was attributed to the micronutrient fraction of the TWD, and restoration of calcium and vitamin D to standard amounts ameliorated the tumor-promoting effects of TWD. Finally, exposure to the TWD elicited large scale, dynamic changes in mRNA signatures of colon mucosa associated with interferon (IFN) response, inflammation, innate immunity, adaptive immunity, and antigen processing pathways, among others. Taken together, these observations indicate that consumption of the TWD markedly enhanced colitis, delayed recovery from gut injury, and enhanced colon tumorigenesis likely via extensive changes in expression of immune-related genes in the colon mucosa.

Extracellular vs Intracellular Delivery of CO: Does It Matter for a Stable, Diffusible Gasotransmitter?

Carbon monoxide (CO) is a gasotransmitter produced in humans. An essential unanswered question in the design of carbon monoxide releasing molecules (CORMs) is whether the delivery molecule should be localized extra- or intracellularly to produce desired biological effects. Herein we show that extracellular CO release is less toxic and is sufficient to produce an anti-inflammatory effect similar to that of intracellular CO release at nanomolar concentrations. This information is valuable for the design of CORMs.